US9017989B2 - Means for reducing acetoin buildup in alcoholic fermentation media - Google Patents

Means for reducing acetoin buildup in alcoholic fermentation media Download PDF

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US9017989B2
US9017989B2 US12/451,339 US45133908A US9017989B2 US 9017989 B2 US9017989 B2 US 9017989B2 US 45133908 A US45133908 A US 45133908A US 9017989 B2 US9017989 B2 US 9017989B2
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bdh1
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acetoin
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Sylvie Dequin
Maryam Ehsani
Maria Rosario Fernández Gallegos
Josep A. Biosca
Anne Ortiz-Julien
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/02—Preparation of other alcoholic beverages by fermentation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G1/00—Preparation of wine or sparkling wine
    • C12G1/02—Preparation of must from grapes; Must treatment and fermentation
    • C12G1/0203—Preparation of must from grapes; Must treatment and fermentation by microbiological or enzymatic treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/08—Preparation of other alcoholic beverages by methods for altering the composition of fermented solutions or alcoholic beverages not provided for in groups C12G3/02 - C12G3/07
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G2200/00—Special features
    • C12G2200/11—Use of genetically modified microorganisms in the preparation of wine

Definitions

  • the invention relates to yeasts and to fermentation methods using these yeasts, for reducing acetoin buildup in alcoholic fermentation media.
  • Saccharomyces cerevisiae yeasts in particular enological Saccharomyces cerevisiae yeasts, convert sugars to alcohol with a yield of 0.47 g/g, which varies little according to the strain used.
  • the production of a Saccharomyces cerevisiae yeast with a low alcohol yield requires the implementation of genetic strategies aimed at diverting a part of the sugars to the formation of other by-products.
  • the solution proposed by the inventors consists in converting the acetoin that is produced into 2,3-butanediol, a compound considered to be neutral from an organoleptic point of view, the detection threshold thereof in wine being greater than 12 g/l.
  • Bdh1p butanediol dehydrogenase
  • the 2,3-butanediol production pathway contributes to the intracellular NAD + /NADH redox balance. In bacteria, this pathway can also be involved in the regulation of intracellular pH.
  • 2,3-Butanediol is produced in several forms: (2R,3R)-2,3-butanediol and (2S,3S)-2,3-butanediol, two optically active forms, (2R,3S)-2,3-butanediol and (2S,3R)-2,3-butanediol, corresponding to the meso forms.
  • the butanediol dehydrogenase encoded by BDH1 is the main enzyme involved in the production of 2,3-butanediol. It is responsible for the formation of all the (2R,3R)-2,3-butanediol and a part of the (meso)-2,3-butanediol, from R-acetoin and S-acetoin, respectively. Since Bdh1p has, moreover, a greater affinity for acetoin (Km acetoin : 4.5 mM, Km 2,3-butanediol : 14 mM), the reaction is strongly shifted in the direction of the formation of 2,3-butanediol.
  • the objective of the invention is therefore to provide novel yeast strains genetically transformed so as to overexpress BDH1 and exhibiting a modified, cofactor specificity, in order to produce larger amounts of 2,3-butanediol from acetoin.
  • the invention is also directed toward providing a method for obtaining such strains.
  • the objective of the invention is also to take advantage of the properties of these transformed yeast strains in a method of alcoholic fermentation and also for the production of 2,3-butanediol.
  • the yeast strains of the invention are yeasts which overexpress, relative to the initial strain, the BDH1 gene encoding Bdh1p.
  • strains are characterized in that they catalyze, in an alcoholic fermentation medium, the reduction of acetoin to 2,3-butanediol according to a rate that is at least twice that of the initial strain.
  • strains that have been genetically transformed and/or mutated in such a way as to obtain an acetoin-to-2,3-butanediol conversion rate that is at least twice that of the initial strain.
  • initial strain is intended to mean a strain before overexpression or modification of the BDH1 gene.
  • the invention is thus directed toward yeast strains as defined above, genetically transformed so as to overexpress BDH1, by means of regulatory sequences suitable for increasing the expression of said gene.
  • Such transformed strains contain at least 2 copies of the BDH1 gene.
  • Any promoter that is active in the host in which it is desired to obtain the expression of this gene may be used, preferably promoters described as strong (encoding strongly expressed genes) under alcoholic fermentation conditions. This is the case, for example, of glycolytic genes strongly expressed in fermentation, such as ADH1 (alcohol dehydrogenase), PGK1 (phosphoglycerate kinase) or TDH3 (glyceraldehyde dehydrogenase), or the TEF1 transcription factor gene, but many others also exist.
  • the invention is also directed toward strains in which the BDH1 gene comprises one or more mutations in such a way as to encode a Bdh1p protein in which one or more amino acids are mutated.
  • such strains comprise one or more mutations in the BDH1 gene in order to exhibit an increased affinity for the NADPH cofactor instead of NADH.
  • the choice of amino acids to be modified is based on the fact that NAD(H) differs from NADP(H) in terms of the phosphate group esterified at the 2′-position of the ribose of adenosine. As a result, the amino acids that interact with this group are candidates for the cofactor specificity change.
  • the residue which determines the NAD(H) specificity is aspartate (Asp) or glutamate (Glu), which form hydrogen bonds with the 2′- and 3′-hydroxyl groups in the ribosyl part of the coenzyme.
  • NADP(H)-dependent dehydrogenases have a smaller and neutral residue, such as glycine (Gly), alanine (Ala) and serine (Ser), at the same position.
  • Gly glycine
  • Al alanine
  • Ser serine
  • an adjacent arginine residue (Arg) enables good interaction with the phosphate group of NADP(H).
  • advantageous replacements in accordance with the invention concern the Glu(E)221 residue replaced with Ser(S), the Ile(I)222 residue replaced with Arg(R) and the Ala(A)223 residue replaced with Ser, using as a basis the structure of the NADPH-dependent Adh6p enzyme of S. cerevisiae (the positions are denoted according to the amino acid sequence of Bdh1p of Saccharomyces cerevisiae S288C).
  • the first residue may also be a serine (as in the example), or any small, neutral amino acid such as Gly or Ala.
  • the invention is also directed toward strains as defined above, genetically modified so as to overexpress BDH1 in an alcoholic fermentation medium, by means of a strong yeast promoter, and comprising one or more mutations in BDH1 in order to exhibit a cofactor specificity for NADPH instead of NADH.
  • the yeast strains which produce acetoin are strains that overproduce glycerol. They are in particular strains containing the overexpressed GPD1 gene or the overexpressed GPD2 gene, for example obtained by transformation of the strains with the pVT100U-ZEO-URA3-GPD1 plasmid or by in situ exchange of its promoter with a strong promoter.
  • the yeast strains are genetically modified in such a way as to reduce the production of acetate (in addition to the overexpression of GPD1). More particularly, they are strains in which the ALD6 gene and, where appropriate, copies thereof has (have) been deleted.
  • Preferred strains belong to the Saccharomyces genus and comprise, in particular, the species Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces uvarum and Saccharomyces kudriavzevii.
  • the invention is also directed toward the hybrids of the strains defined above.
  • the invention is also directed toward a method for obtaining the yeast strains mentioned above.
  • This method advantageously takes advantage of the genetic engineering and site-directed mutagenesis techniques.
  • an oligonucleotide comprising the desired target mutations in order to modify the BDH1 gene of a yeast strain, and the transformation of the strain with an amplified fragment, the amplification of a region of the gene comprising the mutations, or alternatively crossing between strains, starting from a strain comprising, for example, the desired mutations in order to transfer them into another.
  • a subject of the invention is also a method of fermentation, characterized by the addition of a transformed yeast strain as defined above to the fermentation medium.
  • the fermentation medium is in particular a must, advantageously a grape juice.
  • the 2,3-butanediol obtained using the yeast strains of the invention constitutes an important compound for a variety of chemically based materials and liquid fuels.
  • This compound may result, by dehydration, in the formation of methyl ethyl ketone, that can be used as a liquid fuel additive.
  • the 2,3-butanediol may also be converted to 1,3-butanediene, a compound that is used in the production of synthetic gum.
  • Other derivatives for uses, for example, as antifreezes (levo form), solvents and plastics, may also be prepared from 2,3-butanediol.
  • it may be added to food products as a flavor after conversion to diacetyl by dehydrogenation.
  • esterification of butanediol leads to the formation of polyurethane precursors for use in medicaments, cosmetic products and lotions, etc.
  • yeast strains for producing 2,3-butanediol in particular in the applications mentioned above, is also part of the field of the invention.
  • the invention provides means for obtaining a higher 2,3-butanediol yield and productivity in general, but also for obtaining pure stereoisomers, in large amounts, instead of a mixture of isomers.
  • the yeast strains of the invention also make it possible to reduce the amount of diacetyl produced. They are advantageously used to this effect, in particular in fermented beverages.
  • FIGS. 1 to 7 represent, respectively:
  • FIG. 1 the effect of the overexpression of BDH1 on the production of acetoin by the V5ald6 BDH1 pGPD1 strain for varying glycerol concentrations;
  • FIG. 3 sequence alignment of BDH1 (in bold) (SEQ ID NO:10) and BDH1 223 (SEQ ID NO:11).
  • SEQ ID NO:10 sequence alignment of BDH1 (in bold) (SEQ ID NO:10) and BDH1 223 (SEQ ID NO:11).
  • gray the change of 3 amino acid residues E221S/V222R/A223S inducing a reversion of the cofactor specificity in Bdh1p;
  • FIG. 4 acetoin formation in the V5ald6 BDH1 pGPD1 (black) and V5ald6 BDH1 223 pGPD1 (white) strains over the course of a fermentation;
  • FIG. 5 the final formation of acetoin in the CEN.PKald6 pGPD1 (white), CEN.PKald6 BDH1 pGPD1 (gray) and CEN.PKald6 BDH1 223 pGPD1 (black) strains at the end of several fermentations;
  • FIG. 6 the functioning of mutant forms in vivo.
  • FIG. 7 the reduction of diacetyl formation by overexpression of BDH1.
  • the BDH1 promoter was replaced with the TDH3 promoter in situ, using the short flanking homologous (SFH) PCR (polymerase chain reaction) technique (Guldener et al., 1996).
  • SFH short flanking homologous
  • a fragment constituted of the kanMX module, which carries the kanR gene conferring resistance to the G418 R antibiotic, and of the TDH3 promoter was amplified by PCR from the bacterial plasmid pUG6-NOXE (Heux et al., 2005) using the oligonucleotides having the sequences, respectively, SEQ ID No. 1 and SEQ ID No. 2, carrying a sequence homologous to pUG6-NOXE and a flanking sequence (in italics) homologous to the chromosomal target region (BDH1 promoter).
  • SEQ ID No. 1 5′ CTTTCCTCCT TACGGGGTCC TAGCCTGTTT CTCTTGATAT GCAGGTCGAC AACCCTTAAT 3′
  • SEQ ID No. 2 5′ AGTGAATATC ACCCTTCTTG AAATATGCCA AAGCTCTCAT TCGAAACTAA GTTCTTGGTGT 3′
  • DyNazyme EXT 0.75 ⁇ l (Finnzymes, Finland) 30 cycles (45 sec 94° C., 30 sec 55° C., 2 min 72° C.) on a Perkin-Elmer Cetus model 9600 amplifier.
  • the PCR product obtained was precipitated with ethanol in the presence of salts. 3 ⁇ g of the precipitated DNA were used to transform the S. cerevisiae yeast strains V5 and V5ald6.
  • the V5 strain (MATa, ura3) is derived from an enological strain.
  • the V5 strain and the V5ald6 strain (Remize et al., 1999) in which the two copies of the ALD6 gene were deleted were transformed by the lithium acetate method (Schiestl and Gietz, 1989).
  • the transformants were selected on YPD G418 R dishes. The integration of the TDH3 promoter in place of the BDH1 promoter was verified by PCR.
  • the results obtained show that the V5 BDH1 strain has the same growth and fermentation rate as the V5 control strain.
  • the measurement of the BDH activity during the exponential phase and the stationary phase shows that the BDH enzymatic activity of the strain overexpressing BDH1 is approximately 30 times greater than that of the wild-type strain (average specific activity V5:0.1 U/mg total protein; average specific activity V5 BDH1:3.2 U/mg total protein).
  • V5, V5ald6, V5 BDH1 and V5ald6 BDH1 strains were transformed with 10 ng of the multicopy vector pVT100U-ZEO-GPD1 (Remize et al., 1999) carrying the URA3 gene.
  • the transformants were selected on YNB dishes supplemented with methionine (115 mg/l).
  • methionine 115 mg/l.
  • the fermentations were carried out in 1.2-liter reactors (SGI, France) with a reaction volume of 1 liter.
  • the MS medium was used for preculturing and culturing. It is a synthetic medium which simulates a standard grape must (Bely et al., 1990).
  • the MS medium contains 20% glucose, 6 g/l of malic acid, 6 g/l of citric acid, and 460 mg/l of nitrogen, in the form of NH 4 Cl (120 mg/l) and of amino acids (340 mg/l).
  • the medium is supplemented with methionine (115 mg/l) and, if necessary, uracil (50 mg/l).
  • the pH of the MS medium is 3.3.
  • Anaerobiosis factors ergosterol (7.5 mg/l), oleic acid (2.5 mg/l) and Tween 80 (0.21 g/l) are added.
  • the precultures were prepared in 100 ml Erlenmeyer flasks containing 20 ml of medium at 28° C. with shaking (150 rpm) for 30 h.
  • the reactors were inoculated using these precultures, at a cell density of 1 ⁇ 10 6 cells/ml, and maintained at a constant temperature of 28° C. with continuous shaking (300 rpm).
  • the culture samples were collected using a syringe.
  • the fermentation data are expressed as a function of time.
  • Analytical methods The growth was monitored by measuring the optical density at 600 nm and by counting the number of cells on a Coulter Counter instrument (ZBI) using an aliquot fraction of the culture medium.
  • ZBI Coulter Counter instrument
  • the metabolites were assayed in the supernatant, after centrifugation, of the samples taken, at 13 000 rpm for 5 minutes.
  • the glucose, glycerol, ethanol, pyruvate, succinate, acetate, ⁇ -ketoglutarate and 2-hydroxy-glutarate concentrations were determined by high pressure liquid chromatography (HPLC) using an HPX-87H column (Bio-Rad).
  • HPLC high pressure liquid chromatography
  • the acetaldehyde concentration was determined by the enzymatic method described by Lundquist (1974).
  • the acetoin and 2,3-butanediol concentrations were determined by gas chromatography as described by Michnick et al., 1997.
  • FIG. 1 shows the impact of the overexpression of BDH1 on acetoin production.
  • the production of this compound was analyzed over the course of fermentation with V5ald6 pGPD1 and V5ald6 BDH1 pGPD1.
  • Two different conditions of glycerol overproduction were used. Under conditions (i), no selection pressure was used to maintain the pVT100U-ZEO-GPD1 plasmid, resulting in a moderate overproduction of glycerol, reaching 10-15 g/l.
  • V5 BDH1 pGPD1 and V5ald6 BDH1 pGPD1 strains were cultured in the absence of uracil, enabling the plasmid to be maintained throughout the fermentation, generating a very high overproduction of glycerol, 20-30 g/l.
  • the overexpression of BDH1 allows a drastic decrease in acetoin production of up to 90% relative to the level produced by the V5ald6 pGPD1 control strain, and a corresponding increase in the production of 2,3-butanediol ( FIG. 2 ).
  • the V5 strain was used as a control. This strain does not build up acetoin and produces approximately 0.5 g/l of 2,3-butanediol.
  • FIG. 2 shows the acetoin levels obtained after several fermentation experiments with the V5ald6 pGPD1 and V5ald6 BDH1 pGPD1 strains, under the same conditions as above.
  • Wild-type enzyme BDH1 of S288C 221 EIAERR 226 Single mutant: E221S 221 SIAERR 226 Double mutant: E221S/I222R 221 SRAERR 226 Triple mutant: E221S/I222R/A223S 221 SRSERR 226
  • the mutations were introduced using a method based on the Quickchange II XL Site-Directed Mutagenesis Kit
  • the E221S mutant was constructed from the coding region of BDH1 of the laboratory strain S288C cloned into the pYES2 plasmid (Invitrogen, Carlsbad, Calif.): pYES2-BDH1 (E. Gonzalez, 2000).
  • This shuttle vector contains an inducible promoter (promoter and UAS sequence of GAL1), the 2 ⁇ origin of replication, the URA3 selectable marker and the bacterial elements (origin of replication and ampicillin-resistance gene).
  • the pYES2-BDH1 plasmid containing the E221S, E221S/I222R and E221S/I222R/A223S mutations was used as a template to obtain the single mutant, the double mutant and the triple mutant, respectively.
  • the mutagenesis was carried out by PCR using oligonucleotides containing the mutations (in bold) in the forward direction and in the reverse direction (E221S: MUT 221-1 (SEQ ID No. 3), MUT 221-2 (SEQ ID No. 4); E221S/I222R: MUT 222-1 (SEQ ID No. 5), MUT 222-2 (SEQ ID No. 6); E221S/I222R/A223S: MUT 223-1 (SEQ ID No. 7), MUT 223-2 (SEQ ID No. 8)).
  • SEQ ID No. 3 (MUT223221-1) 5′ GGGCTAGTAAAATTGTAGTCT TCA ATTGCAGAGAGAAGAATAGAA ATGG 3′, SEQ ID No. 4 (MUT223221-2) 5′ CCATTTCTATTCTTCTCTCTGCAAT TGA AGACACTACAATTTTACTA GCCCC 3′ SEQ ID No. 5 (MUT224222-1) 5′ GGGGCTAGTAAAATTGTAGTGTCT TCAAGA GCAGAGAGAAGAATAGA AATGG 3′ SEQ ID No. 6 (MUT224222-2) 5′ CCATTTCTATTCTTCTCTCTGC TCTTGA AGACACTACAATTTTACTA GCCCC 3′ SEQ ID No.
  • Escherichia coli cells (ultracompetent XL-10 Gold®, Stratazzo, La Jolla, Calif.) were transformed successively with the constructed plasmids.
  • the transformants were cultured at 37° C. in 5 ml of LB medium supplemented with 50 ⁇ g/ml of ampicillin, and the plasmid DNA of these clones was extracted with the Genelute Plasmid Miniprep® kit (Sigma, USA) and analyzed by sequencing.
  • the laboratory strain FY834 bdh1 ⁇ (MAT ⁇ his3 ⁇ 200 ura3-52 leu2 ⁇ 1 lys2 ⁇ trp1 ⁇ ) was transformed with pYES2-BDH1, pYES2-MUT 221, pYES2-MUT 222, pYES2-MUT 223.
  • the transformation method used is the lithium acetate method described by Schiestl and Gietz (1989).
  • the selective medium used to select the strains transformed with the plasmids is YNB (0.67% yeast nitrogen base, 2% glucose) supplemented with histidine, leucine, lysine and tryptophan.
  • YNB medium 20 ml of YNB medium (20% galactose) were inoculated with 5 transformants, respectively. The culturing was carried out at 28° C. for 2 days.
  • the butanediol dehydrogenase activity was determined using the crude protein extracts at 25° C., by measuring the change in absorbance at 340 nm.
  • One activity unit corresponds to 1 ⁇ mol of cofactor used per min, based on an absorption coefficient of 6220 cm ⁇ 1 M ⁇ 1 at 340 nm for NADH and NADPH.
  • the optical paths for the reduction reactions were 1 cm, 0.5 cm and 0.2 cm.
  • the enzymatic activity assays were carried out in the presence of 50 mM of acetoin in 33 mM NaH 2 PO 4 , pH 7.0/NaOH.
  • the kinetic parameters were obtained by means of assays of activity with coenzyme concentrations of 1 ⁇ 3 ⁇ K m to 10 ⁇ K m of BDH for NADH (0.055 mM).
  • the BDH activity was determined on crude extracts of the strain expressing the wild-type form (pYES2-BDH1) and of the strains expressing the mutated forms.
  • the kinetic parameters of the various forms were also determined (table 2).
  • the three mutants exhibit an affinity for NADH that is very greatly reduced, by 90% on average relative to that of the native enzyme.
  • the data as a whole show that the mutations made very greatly reduce the NADH-dependent activity of the enzyme and introduce an affinity for NADPH.
  • the three mutants exhibit a similar affinity for NADPH.
  • the double-mutant and triple-mutant forms are particularly advantageous because they have a better specific activity compared with the single mutant.
  • the BDH1 gene carrying the triple mutation was overexpressed in this strain by in situ site-directed mutagenesis.
  • Two oligonucleotides, including one of 55 base pairs (SEQ ID No. 9) containing the target mutations, were synthesized and used to amplify the loxpKanMXloxp-TDH3p-BDH1 region of a V5ald6 BDH1 strain.
  • This strategy made it possible, in a single step, to integrate the target mutations into the genomic sequence of Bdh1p, by homologous recombination, while at the same time overexpressing the mutated gene.
  • Oligonucleotide (1) 5 ⁇ l (20 pmol) Oligonucleotide (2) 5 ⁇ l (20 pmol) 10X Taq buffer + 15 mM MgCl 2 5 ⁇ l 2.5 mM dNTP 4 ⁇ l Total V5ald6 BDH1 DNA 2 ⁇ l (50 ng/ ⁇ l) H 2 O 28.25 ⁇ l Taq 0.75 ⁇ l 30 cycles (10 cycles: 20 sec 94° C., 20 sec 60° C., 3 min 72° C.; 20 cycles: 15 sec 94° C., 30 sec 60° C., 2 min + 20 s/cycle 72° C.).
  • the PCR product obtained was precipitated with ethanol in the presence of salts. 4 ⁇ g of the precipitated DNA were used to transform the S. cerevisiae V5ald6 yeast strains.
  • the integration was verified by enzymatic digestion of the PCR product for 2 hours at 37° C. with the Bbs I enzyme using the following reaction mixture: 13.5 ⁇ l of PCR product; 1.5 ⁇ l of 10 ⁇ NEB2 buffer (Biolabs, USA); 0.25 ⁇ l (1.25 U) of Bbs I (Biolabs, USA).
  • the enzymatic activity was then determined on the crude protein extract of a strain having integrated the desired mutations. For each assay, 20 ⁇ l of crude extract were used in the presence of 0.2 mM of NADH/NADPH and 50 mM of acetoin.
  • the modified region was sequenced, thereby making it possible to verify that the mutated BDH1 sequence is identical to the sequence of the native gene, with the exception of the mutations introduced, which result in 3 amino acids being changed in the protein ( FIG. 3 ).
  • V5ald6 BDH1 pGPD1 strain overexpressing wild-type Bdh1
  • V5ald6 BDH1 223 pGPD1 NADPH-dependent Bdh1
  • alpha-ketoglutarate may be linked to the limitation of its reduction to glutamate, a reaction which is catalyzed by NADPH-dependent glutamate dehydrogenase Gdh1p.
  • Gdh1p NADPH-dependent glutamate dehydrogenase
  • the inventors furthermore showed that this effect was also obtained in another genetic background, the S. cerevisiae laboratory strain CEN.PK.
  • the overexpression of BDH 223 in CEN.PK ald6 pGPD1 which produces 1638 mg/l of acetoin on a synthetic must containing 50 g/l of glucose, results in a 68% reduction in acetoin production, whereas overexpression of the native enzyme reduces this production by only 18%.
  • the amount of 2,3-butanediol increases stoichiometrically as the acetoin decreases ( FIG. 5 ).
  • the invention thus provides wild-type and mutated yeast strains which overproduce 2,3-butanediol dehydrogenase, including strains which overproduce glycerol, with controlled production of acetate, which build up 2,3-butanediol in large amount and with a considerably reduced production of acetoin.
  • BDH1 223 overexpression may be envisioned for reducing the acetoin in any modified or unmodified yeast strain that builds up this compound, such as strains which overproduce glycerol.
  • Another example is a strain overexpressing a bacterial NADH oxidase. It has in fact been shown that overexpression of the NOXE gene encoding NADH oxidase of Lactococcus lactis , in the V5 strain, decreases the intracellular NADH content, thereby leading to a decrease in the ethanol yield owing to the limitation of the alcohol dehydrogenase activity.
  • mutants described above which exhibit an increased affinity with respect to NADPH, in particular the double mutant which has the same characteristics as the triple mutant, may also prove to be advantageous for reducing acetoin under physiological conditions.
  • NADPH-dependent Bdh1p in yeast makes it possible to modify the NADP/NADPH cofactor balance and, in this respect, may constitute an advantageous tool in the study of the intracellular oxidoreduction equilibrium.
  • the change in specificity of BDH was carried out in the S288C and V5 strains, although the cofactor binding site differs by one amino acid between these 2 strains.
  • the V5 strain in fact has a valine at position 222, whereas S288C has an isoleucine at the same position.
  • the introduction of the E221S, E221S/I(V)222R and E221S/I222R/A223S mutations proved to be effective and made it possible to obtain a complete change in cofactor specificity from NADH to NADPH.
  • the study shows that the 12 strains analyzed can be divided up into 2 groups, one (DBVPG1373, DBVPG1853, DBVPG6765, L — 1374, L — 1528 and SK1) characterized by the 221 EVA 223 sequence (V5-type sequence), the other (DBVPG6044, S288C, Y55, YGPM, YPS128 and YPS606) by the 221 EIA 223 sequence (S288C-type sequence).
  • amino acid changes by site-directed mutagenesis may be entirely applied to yeast species other than S. cerevisiae.
  • the laboratory strain WV36-405 (Mata, ade2, ura3-52, trp1, adh1 ⁇ , adh2 ⁇ , adh3 ⁇ , adh4:: TRP1) (Atrian et al., 1990) was also transformed with pYes2, pYes2-BDH1, pYes2-MUT 221, pYes2-MUT 222 and pYes2-MUT 223 by the described lithium acetate transformation method (Schiestl & Gietz, 1989).
  • the selective medium used to select the transformed strains is YNB (0.67% yeast nitrogen base, 2% glucose) supplemented with adenine.
  • YNB medium 20% galactose
  • the cells were taken in the growth phase, at OD (600 nm) 2.5.
  • the cells (10 9 ) were suspended in 500 ⁇ l of buffer A (20 mM sodium phosphate (pH 7.0) containing 1% glycerol and 0.5 mM DTT) and ground using glass beads. After centrifugation at 12 000 rpm for 5 min, the supernatant was recovered and used for the enzymatic assays.
  • the 2,3-butanediol dehydrogenase activity was determined using the crude extracts by measuring the change in absorbance at 340 nm as described below.
  • the assays were carried out in the presence of 33 mM of sodium phosphate (pH 7.0), 0.5 mg/l of BSA (bovine serum albumin), of acetoin and of 1 mM NAD(P)H.
  • the protein concentration in the extracts was determined with the Bradford method (Bio-Rad).
  • the K m and V m for NAD(P)H (table 3) were determined in the presence of 50 mM and 450 mM of acetoin for Bdh1 and the Bdh1 mutants, respectively.
  • the mutants all exhibit an NADPH-dependent activity (tables 3 and 4).
  • the affinity of the double-mutant and triple-mutant forms for NADPH (44 ⁇ M) is identical to the affinity of Bdh1 for NADH (45 ⁇ M).
  • the comparison of the specific activities of each enzyme in the presence of 1 mM NAD(P)H shows that the triple-mutant form has a substantially higher activity for NADPH than the double mutant.
  • the laboratory strain ENYpgi1 (MATa, ura3-52, leu2-3, trp1-289, his- ⁇ 1, MAL2-8c, MAL3, SUC3, pgi1::KanMX) (Heux et al., 2008) was transformed with pYes2, pYes2-BDH1, pYes2-MUT 221, pYes2-MUT 222 and pYes2-MUT 223.
  • the transformants were selected on uracil-free minimal medium containing 2% of fructose and 0.05% of glucose.
  • Drop tests were carried out using cells taken in the exponential growth phase at OD 600 1.
  • the cells were serially diluted 10-fold, and 10 ⁇ l of each dilution were deposited on YEP (1% yeast extract, 2% peptone) agar medium with 2% fructose plus 0.05% galactose; YEP agar medium with 0.5% galactose or YEP agar medium with 0.5% galactose plus 0.05%, 0.1% or 0.2% acetoin.
  • the ENYpgi1 strain is deleted of PGI1 which encodes phosphoglucose isomerase. This enzyme is located at the junction of glycolysis and the pentose phosphate pathway (PPP). In this mutant, glucose-6-phosphate is entirely redirected to the PPP, thereby generating an excess of NADPH which prevents the strain from developing. On the other hand, this mutant can develop if it is provided with an NADPH reoxidation system, for example provided by expression of the E. coli transhydrogenase udha.
  • FIG. 6 shows that ENYpgi cannot grow on galactose as the sole carbon source, whereas the expression of udha makes it possible to restore its growth. If acetoin is added to the culture medium ( FIG. 6 c ), the growth of ENYpgi1 expressing the NADPH-dependent mutated forms of Bdh1 is also restored. It follows from these results that the NADPH-dependent BDH forms are capable of reoxidizing the NADPH produced in excess by this strain, to NADP, thereby demonstrating their functionality in vivo.
  • Diacetyl (2,3-butanedione) is undesirable in fermented beverages.
  • its detection threshold ranges between 0.2 and 2.8 mg/l, and it is considered to be undesirable above 5 mg/l.
  • diacetyl poses a major problem owing to its very low detection threshold (0.1 ppm) and a long maturation stage is necessary in order to eliminate this compound.
  • Diacetyl comes from the oxidative decarboxylation of ⁇ -acetolactate, an intermediate in the biosynthesis of isoleucine, leucine and valine, and can be reduced to acetoin. It has been shown that Bdh1 has, in vitro, a diacetyl-reducing activity. In order to study the impact of Bdh1 on diacetyl in vivo, the final concentration of diacetyl produced by the V5ald6 pGPD1 and V5ald6 BDH1 pGPD1 strains in the experiment described in FIG. 2 a was assayed.
  • the diacetyl concentration was determined by SPME (solid-phase microextraction) using deuterated diacetyl-d6 as internal standard, and GC-MS (Hayasaka & Bartowsky, 1999).
  • SPME solid-phase microextraction
  • GC-MS Hexasaka & Bartowsky, 1999.
  • the overexpression of BDH1 makes it possible to reduce the production of diacetyl by a factor of 2 ( FIG. 7 ). Similar results were obtained with a strain overexpressing the triple-mutant form of Bdh1.

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KR101773123B1 (ko) * 2015-01-29 2017-09-12 서울대학교산학협력단 2,3-부탄다이올 생산능을 갖는 유전적으로 조작된 효모 세포 및 그를 사용하여 2,3-부탄다이올을 생산하는 방법
WO2016122107A1 (ko) * 2015-01-29 2016-08-04 서울대학교 산학협력단 2,3-부탄다이올 또는 아세토인 생산능을 갖는 유전적으로 조작된 효모 세포 및 그를 사용하여 2,3-부탄다이올 또는 아세토인을 생산하는 방법
KR101819189B1 (ko) * 2015-12-03 2018-01-16 서울대학교산학협력단 아세토인 생산능을 갖는 유전적으로 조작된 효모 세포 및 그를 사용하여 아세토인을 생산하는 방법
CN113136347B (zh) * 2020-01-20 2022-06-03 暨南大学 高产松柏醇的酿酒酵母工程菌及其构建和应用
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